Chromatographia (2011) 73:781–786 DOI 10.1007/s10337-011-1946-1
FULL SHORT COMMUNICATION
Quantitative Determination of Lobeline Hydrochloride in Rabbit Plasma by LC–MS–MS and Its Application Zhennan Zhang • Gexin Dai • Yaozhen Ye Zhe Sun • Meilfei Lu • Jianshe Ma • Xianqin Wang
•
Received: 2 November 2010 / Revised: 21 January 2011 / Accepted: 27 January 2011 / Published online: 15 February 2011 Ó Springer-Verlag 2011
Abstract A sensitive and selective liquid chromatography tandem mass spectrometry method for quantitative determination of lobeline hydrochloride in rabbit plasma was developed and validated. After addition of triazolam as internal standard, protein precipitation by acetonitrile was used as sample preparation. Chromatographic separation was achieved on a Zorbax SB-C18 column with acetonitrile-0.1% formic acid as mobile phase with gradient elution. Electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring mode was used for quantification using target fragment ions m/z 338.1 ? 315.8 for lobeline hydrochloride and m/z 342.9 ? 308.0 for the IS. Calibration plots were linear over the range of 2–500 ng mL-1 for lobeline hydrochloride in plasma. Lower limit of quantitation for lobeline hydrochloride was 2 ng mL-1. Mean recovery of lobeline hydrochloride from plasma was in the range 97.5–102.3%. RSD of intra-day and inter-day precision were both \9%. This developed method is successfully used in pharmacokinetic study of lobeline hydrochloride in rabbit.
Z. Zhang Y. Ye Z. Sun M. Lu X. Wang (&) Analytical and Testing Center, Wenzhou Medical College, Wenzhou 325035, China e-mail:
[email protected] G. Dai The First Affiliated Hospital, Wenzhou Medical College, Wenzhou 325000, China J. Ma (&) Function Experiment Teaching Center, Wenzhou Medical College, Wenzhou 325035, China e-mail:
[email protected]
Keywords Column liquid chromatography–tandem mass spectrometry Lobeline hydrochloride Pharmacokinetics Plasma
Introduction Lobeline, 2-[6-(2-hydroxy-2-phenyl–ethyl)-1-methyl-2piperidyl]-1-phenyl-ethanone, is a lipophilic, non-pyridino, alkaloidal constituent of Lobelia inflate Linn., also known as Rapuntium inflatum Mill., Indian weed, pukeweed, asthma weed, gagroot, vomitwort, bladderpod, eyebright, and Indian tobacco [1]. It has many nicotine-like effects [2], including tachycardia and hypertension [3], bradycardia and hypotension in anesthetized rats [4], hyperalgesia [5], as well as analgesia after intrathecal, but not after subcutaneous, administration [6], anxiolytic activity [7], and improvement of learning and memory [8]. There have been several methods published for analysis of lobeline [2, 9–15], such as high performance liquid chromatography (HPLC) [15], liquid chromatography–ion trap tandem mass spectrometry (LC–MSn) [2], spectrophotometer [13, 14] and paper chromatography [12]. But there were two methods reported for the analysis of lobeline in biological samples [2, 15]. Song et al. [2] reported a LC–MSn method for analysis of metabolites of lobeline in the rat urine with solid-phase extraction (SPE) as sample preparation. The identification and structural elucidation of the metabolites were performed by comparing their changes in molecular mass, full-scan MSn spectra with those of the parent drug. Ten metabolites of lobeline were found in rat urine. Reavill et al. [15] reported a HPLC method for quantifying nicotine, cytisine and lobeline with protein precipitation by methanol as sample preparation, but the method was not fully validated. To our best knowledge,
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there is no report for quantitative determination of lobeline in biological sample by LC–MS–MS. The mass spectrometry (MS) detection method has a much higher selectivity than ultraviolet, chemiluminescence and electrochemical detector, and can separate analytes from co-eluents based on their mass-to-charge ratios. In this paper, a selective and sensitive LC–MS–MS method for the quantitative determination of lobeline hydrochloride in rabbit plasma using one-step protein precipitation was developed and validated. The LC–MS–MS method was successfully applied to a pharmacokinetic study of lobeline hydrochloride after intravenous administration to rabbits.
Experimental Chemicals and Reagents Lobeline hydrochloride (98%) was purchased from Shanghai Harvest Pharmaceutical Corporation Limited (Shanghai, China) and triazolam methanol solution (1.0 mg mL-1) was purchased from Institute of Forensic Science under the Ministry of Justice (Shanghai, China). LC-grade acetonitrile and methanol were from Merck Company (Darmstadt, Germany). Ultra-pure water was prepared by a Millipore Milli-Q purification system (Bedford, MA, USA). Instrumentation and Conditions All analyses were performed with a 1200 Series liquid chromatograph (Agilent Technologies, Waldbronn, Germany), and a Bruker Esquire HCT ion-trap mass spectrometer (Bruker Technologies, Bremen, Germany) equipped with an electrospray ion source and controlled by ChemStation software (Version B.01.03 [204], Agilent Technologies, Waldbronn, Germany). Chromatographic separation was achieved on an Agilent Zorbax SB-C18 (2.1 mm 9 50 mm, 3.5 lm) column at 40 °C, with acetonitrile-0.1% formic acid as mobile phase. The flow rate was 0.3 mL min-1. A gradient elution programme was conducted for chromatographic separation with mobile phase A (0.1% formic acid in water) and mobile phase B (acetonitrile) as follows: 0–1.5 min (10–85% B), 1.5–6.0 min (85–85% B), 6.0–7.0 min (85–10% B), 7.0–10.0 min (10–10% B). Drying gas flow and nebuliser pressure was set at 6 L min-1 and 20 psi. Dry gas temperature and capillary voltage of the system were adjusted at 350 °C and 3,500 V, respectively. LC–MS–MS was performed with MRM mode using target ions at m/z 338.1 ? 315.8 for lobeline (Fig. 1a) with fragmentation energy of 0.25 v and m/z 342.9 ? 308.0 for IS (Fig. 1b) with fragmentation energy
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Fig. 1 MS–MS product-ion spectrum of lobeline (a) and triazolam (IS, b) with [M?H]? at m/z 338.1 and 342.9 as the precursor ion, respectively
of 0.35 V, in positive ion electrospray ionization interface, respectively. Calibration Standards and Quality Control Samples Individual stock solutions of lobeline hydrochloride (1.0 mg mL-1) and triazolam (internal standard, IS) (500 lg mL-1) were prepared in methanol. Working standard solution (20, 50, 100, 200, 500 ng mL-1, and 1, 2, 5 lg mL-1) for calibration and controls of lobeline hydrochloride were prepared by serial dilution of the stock solution with methanol. 500 ng mL-1 working standard solution of IS was prepared by dilution of the IS stock solution with methanol. All of the solutions were stored at 4 °C and were brought to room temperature before use. Lobeline hydrochloride calibration standards were prepared by spiking blank rabbit plasma with appropriate amounts of the working solutions. Calibration plots were constructed in the range 2–500 ng mL-1 for lobeline hydrochloride in rabbit plasma (concentrations 2, 5, 10, 20, 50, 100, 200 and 500 ng mL-1). Quality-control (QC) samples were prepared by the same way as the calibration standards, three different plasma concentrations (5, 50 and 500 ng mL-1). The analytical standards and QC samples were stored at -20 °C.
Quantitative Determination of Lobeline Hydrochloride
Sample Preparation Before analysis, the plasma sample was thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 10 lL of the internal standard working solution (500 ng mL-1) was added to 100 lL of collected plasma sample followed by the addition of 200 lL acetonitrile. The tubes were vortex mixed for 0.5 min. After centrifugation at 14,900g for 10 min, the supernatant (10 lL) was injected into the LC–MS–MS system for analysis. Method Validation The selectivity of the method was evaluated by analyzing blank rabbit plasma, blank plasma spiked lobeline hydrochloride and IS, and a rabbit plasma sample. Calibration curves were constructed by analyzing spiked calibration samples on three separate days. Peak area ratios of lobeline hydrochloride to IS were plotted against analyte concentrations, and standard curves were well fitted to the equations by linear regression with a weighting factor of the reciprocal of the concentration squared (1/x2) in the concentration range of 2–500 ng mL-1. The LLOQ was estimated in the process of calibration curve construction and was defined as the lowest concentration for which precision (RSD) was better than 20%. To evaluate the matrix effect, blank rabbit plasma was protein precipitated and then spiked with the analyte at 5, 50 and 500 ng mL-1 (six different sources). The corresponding peak areas were then compared to those of neat standard solutions at equivalent concentrations, and this peak area ratio is defined as the matrix effect (ME). The matrix effect of IS was evaluated at the working concentration (50 ng mL-1) in the same manner. Accuracy and precision were assessed by the determination of QC samples at three concentration levels in six replicates (5, 50 and 500 ng mL-1) in three validation days. The precision was expressed by coefficient of variation (RSD) and the accuracy by relative error (RE). The recoveries of lobeline hydrochloride at three QC levels (n = 6) were determined by comparing peak area of the analytes in QC samples to which the analytes were added post-protein precipitation at equivalent concentrations. The recovery of the IS was determined in a similar way. The stabilities of lobeline hydrochloride in rabbit plasma were evaluated by analyzing three replicates of plasma samples at the concentrations of 5 and 500 ng mL-1, which were exposed to different conditions [16]. These results were compared with those obtained for freshly prepared plasma samples. The short-term stability was determined after the exposure of the spiked samples at room temperature for 2 h, and the ready-to-inject samples (after protein precipitation) in the HPLC autosampler at room temperature for 24 h. The
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freeze/thaw stability was evaluated after three complete freeze/thaw cycles (-20 to 25 °C) on consecutive days. The long-term stability was assessed after storage of the standard spiked plasma samples at -20 °C for 30 days. The stability of the IS (50 ng mL-1) was evaluated in a similar way. Pharmacokinetic Study Japanese male rabbits (2.1–2.3 kg) raised from Wenzhou Medical College Laboratory Animal Center (Wenzhou, China). All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Wenzhou Medical College and were in accordance with the Guide for the Care and Use of Laboratory Animals. Animal Ethical Committee License was SYXK (Z2009-0129). Six rabbits were intravenously given with lobeline hydrochloride via marginal ear vein at a dose of 1 mg kg-1 within 1 min. Blood samples (0.3 mL) were collected from the marginal ear vein into heparinized 1.5 mL polythene tubes at 0, 5, 10, 15, 30, 45, 60, 90, 120, 180, 240, 360 min after dosing. The samples were immediately centrifuged at 1,660g for 5 min. The plasma obtained (100 lL) was stored at -20 °C until analysis.
Results and Discussion Method Development The liquid chromatographic conditions were developed to separate as many interference compounds as possible from the analytes. Different columns, such as Zorbax SB-C18 (50 mm 9 2.1 mm, 3.5 lm) and Zorbax Extend-C18 (50 mm 9 2.1 mm, 3.5 lm) were compared for the analysis. A Zorbax SB-C18, 3.5 lm particle column from Agilent demonstrating better selectivity and proper retention for both lobeline and IS was chosen for the separation. The mobile phase played a critical role in achieving good chromatographic behavior (including peak symmetry and short analysis time) and appropriate ionization. Various combinations of acetonitrile, methanol, water and 0.1% formic acid in water with changed content of each component were investigated and compared to identify the optimal mobile phase. Acetonitrile was chosen as the organic solvent because of its suitable sharper peak shape, lower pressure and more stable compared to methanol. Formic acid added into the mobile phase could improve the sensitivity, therefore acetonitrile-0.1% formic acid was chosen as mobile phase. Gradient elution provided better peak symmetry, proper retention time, and avoided the matrix effects for the analyte and IS compared to isocratic elution. Plasma protein precipitation was used for pretreatment of rabbit plasma samples as it is rapid, widely used and
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has a high recovery. The supernatant was directly injected into the LC–MS–MS system for analysis. Methanol, acetonitrile and 10% trichloroacetic acid, were investigated as precipitation reagents, and the recoveries were 70.3, 96.7 and 64.3% for lobeline hydrochloride (100 ng mL-1), respectively. Acetonitrile proved to be the best reagent in terms of the peak shape obtained by LC–MS–MS. It is more simple and rapid than SPE method described in literature [2]. The IS was also chosen during the process of method development. To find a suitable IS, several compounds (including lidocaine, carbamazepine, bupivacaine, triazolam and diazepam) were tested. Triazolam was suitable because of its stable ionization in positive-ion ESI mode and suitable retention time. Selectivity and Matrix Effect Figure 2 shows the typical chromatograms of a blank plasma sample, a blank plasma sample spiked with lobeline
Fig. 2 Representative LC–MS–MS chromatograms of lobeline hydrochloride (1) and triazolam (IS, 2), monitored ions m/z 338.1 ? 315.8 for lobeline, m/z 342.9 ? 308.0 for IS. a blank plasma; b blank plasma spiked with lobeline hydrochloride
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hydrochloride and IS, and a plasma sample. No interfering endogenous substances were observed at the retention times of the analyte and IS. The ME for lobeline hydrochloride at concentrations of 5, 50 and 500 ng mL-1 were measured to be 93.1, 94.2 and 91.7% (n = 6), respectively. The ME for IS (50 ng mL-1) was 95.8% (n = 6). As a result, ME from plasma was negligible in this method. Calibration Curve and Sensitivity The linear regressions of the peak area ratios versus concentrations were fitted over the concentration range 2–500 ng mL-1 for lobeline hydrochloride in rabbit plasma. The calibration data are presented in Table 1. The LLOQ for the determination of lobeline hydrochloride in plasma was 2 ng mL-1. The precision and accuracy at LLOQ were 10.2 and 93.1%, respectively. The LOD, defined as a signal–noise ratio of 3, was 0.6 ng mL-1 for lobeline hydrochloride in plasma.
(5 ng mL-1) and IS (50 ng mL-1); c a rabbit plasma sample 60 min after intravenous administration of single dosage 1 mg kg-1 lobeline hydrochloride
Quantitative Determination of Lobeline Hydrochloride
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Table 1 Precision, accuracy, recovery and calibration data for lobeline hydrochloride in rabbit plasma (n = 6) Concentration (ng mL-1)
RSD (%)
RE (%)
Intra-day
Inter-day
Recovery (%)
Intra-day
Inter-day
5
7.6
8.5
-6.5
-7.4
98.6
50
2.8
3.7
-4.6
1.4
102.3
500
4.0
1.6
-3.2
2.3
97.5
Linear range (ng mL-1) Slope (mean ± SD) Intercept (mean ± SD) Correlation coefficient (mean ± SD) LOD (ng mL-1) LLOQ (ng mL-1) 2–500
0.06979 ± 0.01102 -0.8032 ± 0.2642
0.9969 ± 0.0012
Table 2 Summary of stability of lobeline hydrochloride and IS under various storage conditions (n = 3) Condition
Concentration (ng mL-1) Added
Ambient, 2 h
5 500
-20 °C, 30 days
50 (IS) 5 500 50 (IS)
3 freeze thaw
5 500 50 (IS)
Autosampler ambient 24 h 5 500 50 (IS)
Found 4.8
RSD RE (%) (%)
0.6
2.0
sample solvent on autosampler was also stable over a 24-h period. The results of stability experiments are listed in Table 2. System Suitability
4.3
-4.0
508.3
1.2
1.7
51.2 4.6
2.2 6.8
2.4 -8.0
492.1
3.2
-1.6
46.7
5.4
-6.6
5.2
9.2
4.0
489.5
4.3
-2.1
48.2
4.7
-3.6
5.1
5.3
2.0
486.3
3.2
-2.7
51.2
4.3
2.4
Precision, Accuracy and Recovery The precision of the method was determined by calculating RSD for QCs at three concentration levels over three validation days. Intra-day precision was 9% or less and the inter-day precision was 8% or less at each QC level. The accuracy of the method was ranged from 92.6 to 102.3% at each QC level. Mean recoveries of lobeline hydrochloride were better than 97.5%. The recovery of the IS (50 ng mL-1) was 98.6%. Assay performance data are presented in Table 1. The above results demonstrate that the values are within the acceptable range and the method is accurate and precise. Stability The stability results showed that lobeline hydrochloride spiked into rabbit plasma was stable for 2 h at room temperature, for 30 days at -20 °C, and during three freeze–thaw cycles. Lobeline hydrochloride extracts in the
Chromatographic parameters calculated from experimental data were given as follow (n = 6): capacity factors (k), 6.2 ± 0.3 for lobeline hydrochloride, 6.5 ± 0.2 for IS; tailing factors, 1.25 ± 0.12 for lobeline hydrochloride, 1.12 ± 0.11 for IS; number of theoretical plates, 5,952 ± 12 for lobeline hydrochloride, 17,273 ± 23 for IS; % RSD of peak area, 4.3 for lobeline hydrochloride, 3.6 for IS; resolution factors (Rs) for lobeline hydrochloride and 1.4 ± 0.1. Application The developed method was applied to a pharmacokinetic study in Japanese male rabbits. The pharmacokinetic data for lobeline were computed from the plasma concentration– time data using a two-compartment model. The main pharmacokinetic parameters were: the area under the plasma concentration–time curve from 0 to t min (AUC0?t) was 7,244.4 ± 1,998.4 min ng mL-1, the area under the plasma concentration–time curve from 0 to infinite min (AUC0??) was 7,364.4 ± 2,095.8 min ng mL-1, the plasma clearance (CL) was 0.15 ± 0.05 L min-1 kg-1, elimination half life (t1/2) was 65.1 ± 16.5 min.
Conclusion A sensitive, simple and specific LC–MS–MS method for quantitative determination of lobeline hydrochloride in rabbit plasma was developed and validated over the concentration range of 2–500 ng mL-1. The simple and rapid protein precipitation by acetonitrile was used for pretreatment of plasma samples. The LC–MS–MS method was successfully applied to a pharmacokinetic study of lobeline
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hydrochloride after intravenous administration of single dosage 1 mg kg-1 to rabbits.
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